Project Summary The long-term goals of this project are to establish germline stem cell (GSC)-based gene editing to generate large animal models of human diseases and for regenerative medicine (PAR-16-093), and to provide accessible systems to study the GSC niche in non-rodent animals. The testis stem cell is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Pigs are important models for pre-clinical research because they are phylogenetically and physiologically more similar to humans than rodents. Gene editing through GSCs rather than embryos will shorten the time necessary to produce germline gene edited animal models. It is also more broadly applicable to disease models where gene dosage and epigenetics have a role and smaller strains of pigs are required. The aims of this renewal project are 1) to establish a culture system that promotes porcine germ cell expansion in vitro; 2) to explore the role of testicular somatic cells in formation of a functional GSC niche; and 3) to develop a precise approach for targeted gene editing in porcine GSCs. While germline transmission of a genetic modification is possible with transplantation of primary pGSCs, efficiency of the approach would benefit from a robust in vitro system for expansion of edited cells prior to transplantation. To accomplish this goal, we will define the metabolic phenotype of pGSCs to inform design of appropriate culture conditions. Grafting to mouse hosts and transplantation to recipient pigs will monitor the ability of cultured cells to support spermatogenesis as functional endpoint. Germ cell function is controlled by interaction with the niche microenvironment. Our novel testicular organoid system and xenografting of testis cells will enable introduction of cell type-specific modifications and characterize stem cell-niche interactions. We will test the hypothesis that primary cilia on testicular somatic cells are essential for niche formation, and that the somatic environment modulates germ cell differentiation and supports germ lineage differentiation of porcine induced pluripotent stem cells (piPSCs). Finally, we will test the hypothesis that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated homology directed repair (HDR) will allow efficient targeted genome editing in GSCs for precise replication of human disease alleles. As HDR is limited to small DNA alterations (~1-50 bp), we will also explore Precise Integration into Target Chromosome (PITCh) for targeted gene editing to enable introduction of large transgenes and/or humanizing parts of the pig genome for regenerative medicine. Introduction of a mutant porcine insulin gene (INSC94Y) that matches the human INSC96Y mutation present in permanent neonatal diabetes mellitus will provide proof-of-principle that our novel strategies are feasible and efficient. Overall, this work will establish precise targeted germline gene editing in pigs, and further define stem cell-niche interactions.